Background/Purpose: mTOR activity is increased in SLE T cells and its blockade has therapeutic efficacy in SLE. Murine studies showed essential roles of mTORC1 in Th1/Th17 and mTORC2 in Th2 differentiation, whereas mTORC1 and 2 need to be blocked for Treg differentiation. Whether mTOR regulates T cell cytokine expression and Treg development in SLE remains elusive.

Methods: CD3⁺ T cells were isolated from SLE patients and matched healthy controls (HC). A part of the CD3⁺ T cells were stained with pS6RP, FoxP3, IFN-γ, IL-4, and IL-17 alone or together following CD4, CD8, and CD25 staining. The rest of the cells were cultured in RPMI culture media with 10% FCS, 1% Penicillin/Streptomycin, and 1% L-glutamine for 3 days in the absence or presence of plate bound anti-CD3 and soluble anti-CD28 with or without 100 nM rapamycin. After 3 day culture, cells were stained as previously described. pS6RP and cytokine expression by CD4⁺, CD8⁺, and DN T cells and frequency of CD4⁺CD25⁺FoxP3⁺ Treg were assessed by flow cytometric analysis. Using day 3 T cell lysates, immunoblotting was performed using anti-Akt, anti-pAkt (Ser 473), anti-S6K1, and anti-pS6K1. The signal intensity was normalized to Actin.

Results: On day 0, frequency of pS6RP^hi cells was higher in DN T cells than CD4⁺ or CD8⁺ T cells (DN T cells: 29.40±6.37%, CD4⁺ T cells: 1.04±0.16%; p=0.00028, CD8⁺ T cells: 5.02±0.73%; p=0.00097 in SLE) and was higher in SLE than HC T cells, which was most prominent in DN T cells (29.40±6.37%, 17.59±4.47%, p<0.001). Rapamycin inhibited mTORC1 in T cells stimulated in vitro for 3 days based on the frequency of pS6RP^hi cells (41.85±7.67%, 1.53±0.27% in SLE; p<0.0001) and pS6K1 expression in T cell lysates (p=0.002) while it augmented mTORC2 based on pAkt expression (p=0.02). On day 0, SLE T cells had higher frequency of IL-4⁺ cells, most prominently in CD8⁺ T cells (7.72±2.40%, 4.00±0.45%, p=0.027). After 3 day in vitro stimulation, DN T cells had higher frequency of IL-4⁺ cells than CD4⁺ or CD8⁺ T cells (DN T cells: 9.89±1.85%, CD4⁺ T cells: 3.08±0.63%; p=0.0018, CD8⁺ T cells: 4.94±0.81%; p=0.014 in SLE). Rapamycin suppressed T cell IL-4 expression, most robustly in DN T cells (9.89±1.85%, 4.92±0.80%, p=0.001). Frequency of IFN-γ⁺ cells was reduced in SLE (p<0.0001). Rapamycin did not suppress T cell IFN-γ expression. After 3 day in vitro stimulation, SLE T cells had higher frequency of IL-17⁺ cells, which was most prominent in CD4⁺ T cells (5.31±1.57%, 2.71±0.63%, p=0.0196) and was suppressed by rapamycin (5.31±1.57%, 2.96±1.12%, p=0.0097). Frequency of Treg was reduced in SLE. Rapamycin blocked mTOR in Treg (pS6RP^hi cells: 45.13±10.97%, 3.39±0.38% in SLE; p<0.001) and promoted its expansion (11.49±1.92%, 19.60±2.80% in SLE; p<0.0001). Neutralization of IL-17 expanded Tregs (p<0.05).  

Conclusion: The data indicate the pathogenic relevance of IL-4 and IL-17 in SLE, which are mainly produced by DN T and CD4⁺ T cells respectively in an mTORC1-dependent manner. In contrast, IFN-γ is conceivably protective against SLE. Rapamycin expands Tregs by blocking mTOR in the Treg and suppressing IL-17. Our study reveals three distinct mechanisms of action by rapamycin in SLE: 1) suppression of DN T cell IL-4 production, 2) suppression of CD4⁺ T cell IL-17 production, and 3) Treg expansion.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mammalian-target-of-rapamycin-mtor-skews-t-cell-lineage-development-in-systemic-lupus-erythematosus-sle/